The effect of obesity and metformin on mouse endometrial proliferation was evaluated in formalin-fixed, paraffin embedded tissue by Ki67 staining (BD Biosciences, San Diego, CA) following the manufacturer’s instructions. The degree of apoptosis in endometrial tissues was determined by IHC staining for cleaved caspase-3 (Asp175; Cell Signaling, Danvers, MA). The sections were counterstained with Mayer’s hematoxylin. The average number of positively-stained Ki67 or Caspase-3 cells in 5–10 high-power microscopic fields were counted per slide, and calculated as: 200×(number of positively stained endometrial cells/total endometrial cells). Tissues were also stained for downstream markers of insulin and IGF1 receptor signaling, including Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-AKT (Ser473), Phospho-p44/42 MAPK (Thr202/Tyr204), and Phospho-Acetyl-CoA Carboxylase (Ser79) (Cell Signaling, Danvers, MA) and scored according to previously published procedures[24 (link)].